The isolation of nucleic acids from agarose or polyacrylamide gels is a routine concern for biological labs. The methods of isolation have taken on greater importance as the study of small DNA and RNA molecules such as micro RNA (miRNA) molecules and small interfering RNA (siRNA) molecules has increased.
Traditional methods for the extraction of DNA from a gel, such as an agarose or polyacrylamide gel, are time-consuming, low-yielding or limited in their application. One method for purifying and extracting oligonucleotides from a gel is the “crush-and-soak” method. For example, a technician will remove the portion of gel containing DNA and crush it in a microcentrifuge tube using a plastic pipette tip, and incubate with constant shaking in an elution buffer (with a high salt concentration) at an elevated temperature. The gel pieces are then eliminated by centrifugation or by passing the mixture through a plug of siliconized glass wool. Finally, DNA is recovered by ethanol precipitation. The process takes several hours, requires utilizing a desalting column and typically retains only half of the desired product.
Another method of extraction is the use of dialysis tubing. The portion of the gel with the desired oligonucleotide product is placed in dialysis tubing with electrophoresis buffer, sealed and placed into an electrophoresis chamber. Applying an electric current will cause the oligonucleotide to migrate out of the gel, but it will be trapped within the bag. When the oligonucleotide is out of the gel, the flow of current is reversed for a few seconds to remove the oligonucleotide from the side of the tubing. The buffer containing the oligonucleotide is then collected and the oligonucleotide is precipitated with ethanol. This method is primarily reserved for the recovery of large (>5 kb) DNA fragments, and is even more time-consuming than the crush-and-soak method.
Oligonucleotides can also be recovered from a gel by use of certain types of silica gel particles. However, small (<100 bp) fragments of DNA are very difficult to elute from standard glass particles.
In the case of miRNAs, the isolation can be difficult. MiRNAs are small non-coding RNAs that are involved in post-transcriptional gene regulation (cf., Bartel, 2004). First identified in C. elegans just over a decade ago (Lee et al., 1993), miRNAs have been identified in virtually every metazoan and plant species examined Experimental evidence is rapidly accumulating that shows miRNAs to play key roles in processes such as cellular differentiation, cell death, and adipose storage as well as in disease processes such as cancer. Over the past few years, a number of investigators have reported on methods for cloning miRNAs from primary RNA sources (Elbashir et al., 2001; Lau et al., 2001; Pfeffer et al., 2003; Sunkar and Zhu, 2004.
The mass of non-coding small RNAs relative to the total RNA produced by a cell is vanishingly small. Thus, the process of isolating and cloning these RNAs (miRNAs are 21 nt to 23 nt in length) is critically dependent upon being able to both enrich and efficiently recover RNA species in the proper size range. One protocol that was developed involves two PAGE gel steps, each of which involves a crush and soak RNA purification from a gel slice and a subsequent desalting via a NAP-5 purification column (Devor et al., U.S. patent application Ser. No. 12/165,436). The desired RNA species at the outset has a low mass, and there is unavoidable loss of mass that accompanies the above purifications.
The current invention extracts the target oligonucleotide rapidly, thereby avoiding burn-off against the positive electrode, yielding high optical density and purity. The need for an osmotic membrane or heavy salt/light salt barrier to capture the oligonucleotide is eliminated. The invention does not require a high salt concentration and therefore does not require a desalting column that is time-consuming and reduces yield. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.